7-Halophenylthioacetamido cephalosporins

ABSTRACT

Certain halogenated phenylthioacetamido cephalosporanic acids and derivatives thereof, e.g., 7-(2&#39;&#39;-(2&#39;&#39;&#39;&#39;,5&#39;&#39;&#39;&#39;dichlorophenylthio)acetamido)cephalosporanic acid, are effective antibiotics against Staphylococcus aureus cultures which show heterogeneous resistance to methicillin.

United States Patent Huffman Sept. 23, 1975 [54]7-HALOPHENYLTHIOACETAMIDO 3,516,997 6/1970 Tarano et a1 260/243 CCEPHALQSPORINS 3,647,789 3/1972 Crast 260/243 C 3,657,232 4/1972 Lemieuxet a1. 260/243 C Inventorr George Huffman, Carmel, 3,663,540 5/1972Lemieux et a1. 260/243 c Assigneez Eli Lilly and p y Indianapolis,3,766,176 10/1973 Lemieux et a1. 260/243 C Ind.

Mar. EXZZMiIleF-NlChOlfiS RlZZO Attorney, Agent, or FirmWilliam C.Martens, Jr.; pp 341,210 Everet F. Smith Related US. Application Data[63] Continuation-impart of Ser. No. 288,227, Sept. 11,

1972, abandoned, which is a continuation-in-part of [57] ABSTRACT Ser.No. 212,739, Dec. 27, 1971, abandoned.

Certain halogenated phenylthioacetamido cephalospo- 52 u.s. c1. 260/243(3; 424/246 mic acids and derivatives thereof, [51 Int. Cl. C07D 501/20dichlorophenylthio)acetamidolcephalosporanic acid, 58 1 Field of Search260/243 c are effective antibiotics against smphylowccus cultures whichshow heterogeneous resistance to [56] References Cited methwillin- N UlTED STATES PATENTS 12 Claims N0 Drawings 3,335,136 8/1967 Flynn 260/243C 7-HALOPHENYLTHIOACETAMIDO CEPHALOSPORINS CROSS REFERENCE This is acontinuation-in-part of application Ser. No. 288,227, filed Sept. ll,1972, and now abandoned, which is a continuation-in-part of applicationSer. No. 212,739. filed Dec. 27, 1971, and now abandoned.

INTRODUCTION This invention relates to cephalosporin antibiotics anduses of such compounds in therapy against diseases caused by methicillinresistant Staphylococcus aureus microorganisms. More particularly thisinvention provides a method for treating and inhibiting the growth ofmethicillin resistant Staphylococcus microorganisms with certainhalogenated phenylthioacetamidocephalosporanic acids and derivativesthereof. Some of such compounds are new.

BACKGROUND OF THE INVENTION a. MethicillinResistant Staphylococcusaureus Various technical journal articles have been written in the pastseveral years describing the effects or the lack of effectiveness ofvarious antibiotics on various methicillin-resistant cultures ofStaphylococcus aureus. These cultures have not occurred too often.However, thei'nfections caused by methicillimresistant strains ofStaphylococcus aureus are said to be nosocomial in nature in that theyappearmostly in bed-ridden or debilitated patients. These pathogenicorganisms have been observed in cultures taken from patients with malignancy, chronic bone and/or joint disease, chronically impairedcirculation or consciousness, or chronic pulmonary disease.

In addition, bacteriologists have been searching for culture preparationrnethods for separately identifying methicillin resistant frommethicillin susceptible cultures of Staphylococcus aureus. Theyapparently have foundat least two i'n'vitro methods, one involvingdifferent temperatures of incubation and one involving the use ofdifferent sodium chloride concentrations in the culture growth medium,which methods help to distinguish methicillin resistant strains ofStaphylococcus aureus from those which are not. With these tools thebacte'riologists can now more effectively assist and advis'e' cliniciansto identify and treat these methicillinresistan tStaphylococcus aur eusconditions. b. Cephalosporin Antibiotic History i CephalosporinC,obtained by fermentation, has been defined as having the followingstructure:

COOH

known as 7 (5 aminoadipamido)cephalosporanic acid. It haswealgantibiotic activity, but it is important as a source of.cephalosporin C nucleus, i.e., 7-am i nocephalosporanic acid (7-ACA),having the structural formula I shown here in zwitterionic form,although anionic and cationic salts may be formed and used. Antibioticssuch as cephalothin and cephaloridine are prepared from 7-ACA by knownmethods. Various derivatives of 7-ACA based antibiotics are made byacylating the 7- amino group of 7-ACA with appropriate acyl acids,halides, or other reactive form of such acyl groups and/or by replacingthe acetoxy group attached to the 3- methyl carbon atom with appropriatenucleophilic groups now well documented in the literature.

In continued research, desacetoxycephalosporin compounds, i.e.,compounds of the structure S H N- H-CH CH (W) c t t c cu II t. y

COOH

In US. Pat. No. 3,335,l36, Flynn disclosed and claimed some halophenylmercaptomethyl cephalosporin compounds which were characterized bypenicilli- 'nase-resistance, acid stability, and activity against bothGram positive and a number of Gram negative organisms. However, thatpatent was silent as to the problems of identifying, and methods fortreating methicillin-resistant Staphylococcus uureus microorganisms.

Furthermore, the patent is silent as to the choice of compounds thatmight be used to treat conditions caused by methicillin-resistantSlapltvlococcus' aurcus microorganisms.

There is a need for finding the most effective antibiotic compounds fortreating methicillin-resistant Staphylococcus aureus microorganism anddisease conditions caused thereby.

It is an object of this invention to provide a method for treatingmethicillin-resistant Staphylacoccus aureus microorganisms with certainpolyhalophenylthioacetamidocephalosporin compounds.

It is a more specific object of this invention to provide some specificmono-chloro-, polychloro, andchlorofluoro-phenylthioacetamidoccphalosporanic acids, or derivativesthereof as being particularly effective in combatting infections causedbymethicillin-resistant Staphylococcus aureus microorganisms, some ofthese compounds being new.

Other objects, aspects and advantages of this invention will becomeapparent from reading the remaining specification.

SUMMARY OF THE INVENTION This invention provides a method for treatingand inhibiting the growth of methicillin-resistant Staphylococcus aureusorganisms and disease conditions caused thereby involving treatment ofthose organisms with certain halophenylthioacetamido cephalosporinderivatives. This invention also provides certain halogenatedphenylthioacetamido cephalosporanic acids and derivatives thereof,effective as antibiotics against Staphylococcus aureus organisms whichexhibit resistance to inethicillin. Most of these compounds are usefulin depot type parenteral administration procedures. At least one ofthese compounds, 7-[2-(3"-chloro-4fluorophenyl)thioacetamido]cephalosporanic acid, so dium salt is new,water soluble and is easily administered by intravenous, andintramuscular injection methods.

DETAILED DESCRIPTION OF THE INVENTION This invention provides animproved method for treating and inhibiting the growth ofmethicillinresistant Staphylococcus microorganisms which comprisestreating the microorganisms with an effective amount of a compound ofthe formula wherein Z is hydrogen or fluorine; and, when Z is hydrogen,each of X and Y is hydrogen or chlorine selected so that the phenyl ringis substituted with l or 2 chlorine atoms and so that when one chlorineatom is present said chlorine atom is in the 3-position, and

when two chlorine atoms are present said chlorine atoms are in the 3,4-,the 3,5- or the 2,5-positions; and

when Z is fluorine, said fluorine is in the 3- or 4- position of thephenyl ring, and each of X and Y is hydrogen or chlorine selected sothat when the phenyl ring is substituted with l or 2 chlorine atoms, oneof the chlorine atoms is in the 3- or 4-position of the phenyl ring;

R is selected from the group consisting of" hydrogen,

C, to C -alkanoyloxy, e.g'., acetoxy,

S-methyl-l ,3,4-thiadiazol-2ylthio,

l-methyll H-tetrazol-S-ylthio lH-tetrazol-5ylthio S-phenyll,3,4-oxadiazol-2-ylthio, S-(p-nitrophenyl )-1 ,3,4-oxadiazol-2-ylthio, 55-(pmethoxyphenyl)-l ,3,4-oxadiazol-2-ylthio,

carbamoyloxy [-O C(O)NH methylcarbamoyloxy, thiomethyl, N-pyridino,azido, dithiocarbamoyl; and R is hydrogen, dicyclohexylamine, or apharmaceutically acceptable cation.

Another aspect of this invention relates to cephalosporin antibioticcompounds of the formula R is hydrogen, dicyclohexylamine, or apharmaceutically acceptable cation;

R is selected from the group consisting of hydrogen, C to C-alkanoyloxy, 5-methyll ,3,4-thiadiazol- 2-ylthio, l-methyllH-tetrazol-S-ylthio, lH-tetrazol- 5-ylthio,S-phenyl-l,3,4-oxadiazol-2-ylthio, 5-(p-nitrophenyl l,3,4-oXadiazol-2-ylthio, 5-(p-methoxyphenyl)-l,3,4-oXadiazol-2-ylthio,carbamoyloxy, methylearbamoyloxy, thiomethyl, N-pyridino, azido, anddithiocarbamoyl;

Z is hydrogen or fluorine; and,

when R is other than C to C -alkanoyloxy, and Z is hydrogen, each of Xand Y is hydrogen or chlorine selected so that the phenyl ring issubstituted with l or 2 chlorine atoms and so that when one chlorineatom is present said chlorine atom is in the 3-position, and when twochlorine atoms are present said chlorine atoms are in the 3,4-, the3,5-, or the 2,5-positions; and,

when R is other than C to C alkanoyloxy and Z is fluorine, said fluorineis in the 3- or 4-position of the phenyl ring, and each of X and Y ishydrogen or chlorine selected so that when the phenyl ring issubstituted with 1 or 2 chlorine atoms, one of the chlorine atoms is inthe 3- or 4-position of the phenyl ring; and,

when R is C to C -alkanoyloxy, and Z is hydrogen, X and Y are chlorineand are in the 3,4-, the 3,5-, or the 2,5-positions; and,

when R is C to C -alkanoyloxy and Z is fluorine, said fluorine is in the4-position of the phenyl ring, and each of X and Y is hydrogen orchlorine selected so that when the phenyl ring is substituted with 1 or2 chlorine atoms, one of the chlorine atoms is in the 3-position of thephenyl ring.

The phenyl ring which is delineated by the X, Y and Z substituentsincludes the following halophenyl moieties: 3-chlorophenyl,3,4-dichlorophenyl, 3,5- dichlorophenyl, 2,5-dichlorophenyl,3-fluorophenyl, 4-fluorophenyl, 3-fluoro-4-chlorophenyl, 3-chloro-4- 5fluorophenyl, 2,4-dichloro-3-fluorophenyl, 3,4-

dichloro-S-fluorophenyl, 2,4-dichloro-5-fluorophenyl,

3,5-dichloro-4-fluorophenyl, 2,5-dichloro-4- fluorophenyl, and the like.

Besides being characterized by penicillinase resis tance, acidstability, and activity against a broad range of Gram positive and anumber of Gram negative pathogens, it has been found that the abovecompounds are highly effective as antibiotics against variousmethicillin-resistant Staphylococcus aureus microorganisms. They areconveniently prepared and administered in the form of the salts of thecarboxyl group with pharmaceutically acceptable cations including, forexample, sodium, potassium, lithium, ammonium, and substituted ammoniumsalts such as methylammonium, ethylammonium, as well as the less watersoluble salts such as the calcium, barium, procaine, quinine,cyclohexylbis(methylamine) and dibenzylethylenediamine salts. Inisolating the compound from its reaction mixture and testing in animalsthe biscyclohexylamine salts are sometimes used. Administration ispreferably by intramuscular injection in sterile water or isotonicsaline or dextrose at a dose (for adults) around 0.25 to 0.5 g. every 4to 6 hours. Oral administration of those compounds which can be absorbedinto the blood by this route generally requires a somewhat higherdosage, from 0.50 to 1.0 g. every 4 to 6 hours, and can be accomplishedin the form of pressed tablet, filled gelatin capsules, suspensions ofconventional type, or the like.

It has been found that most of the compounds of the above describedtype, examples of which are given below, have antibiotic activity[minimum inhibitory concentration (MIC) values] of less than I 1micrograms/- milliliter in the absence of human serum in a standardgradient plate testing procedure against methicillinresistantStaphylococcus aureus. The method used is essentially that described byBryson and Szybalski in 1952 (Science 1 l6:4546). The inoculum treatmentmethod used for penicillin resistant Staphylococci was reported byGodzeski et al. in Antimicrobial Agents and Chemotherapy, May, 1969, pp.547-554.

A standard Falcon square plastic petri dish is used. A layer of agar (10ml.) is poured into one of the square dishes, the dish tipped mm. offthe horizontal, and the agar layer allowed to harden in this position.This bottom layer of agar contains the antibiotic, the serum, and/orother material to be tested. The medium is Difco Penassay Agarcontaining 2 percent agar. We routinely used 4 ml. of human or horseserum for serum-inactivation testing. Serum may be added to the toplayer also so that no serum gradient will exist, but after testing manyantibiotics in this manner no significant differences were noted in thefinal results between the two types of serum plates. Therefore, serum isroutinely added only to the bottom layer.

After the bottom, slanted layer of agar medium has hardened, the plateis placed flat and another ml. of

medium added and allowed to harden.

The inoculum of resistant Staphylococci, as de- .scribed, is prepared bydiluting the water suspension 1/50 in 0.25 percent agar in sterile wateror saline. For Gram negative organisms, an overnight broth culture isdiluted H50 in the 0.25 percent agar-water. This dilu tion is thoroughlyshaken to evenly suspend the bacteria. To streak the plate, we use a 1ml. pipet containing an aliquot of the final 0.05 percent agar dilution.After a little practice, a smooth even stroke of inoculum can be laiddown very rapidly. The trick is in the smoothness of the streak it mustbe accomplished uniformly and quickly.

After 24 hours incubation at 35-37C. the plates are read by simplymeasuring the length of bacterial growth as a percentage of the entirestreak distance which is then converted to the percent of antibioticconcentration in the plate. We have photographically reduced a mm. ruleto just fit inside the dish then the measure in millimeters is equal tothe percent concentration.

Average values of duplicate measurements are calculated and the M.I.C.sreported are averages of 2 streaks on the plate and from 2 to 3duplicate plates. Eight streaks can be easily placed on each plate. Ifsharp end points are not obtained, the middle concentration between thebeginning of inhibition of the streak to the end of growth is measured.The concentration of the antibiotic under examination is varied fromplate to plate in a series as follows:

200 ,ug/ml. Plate No. l and 2 I00 No. 3 and 4 50 No. 5 and 6 20 No. 7and 8 l0 No. 9 and 10 l No. ll and 12 This method of screeningpenicillins and cephalosporins is quite satisfactory. Control platescontaining penicillin and staphcillin or prostaphlin, or other controlantibiotic, are used daily for the resistant staphylococcal activityscreen. The gradient plate method has shown differences betweenantibiotics that were not apparent by any other methods of screening.The antibiotic differences, when examined more closely by other methods,e.g., animal therapy, have proved cor rect. We believe that the gradientplate screen is more sensitive to small differences between relatedantibiot ics than other methods and pictures of the plates can provide apermanent record. The method also lends itself readily to modificationof specific purposes e.g., cross gradients.

The compounds listed below are examples of compounds useful in themethod of this invention. After the compound name there is given inparentheses, where tested, the minimum inhibitory concentrations (MIC)in micrograms per milliliter (,ug/ml) for the compound in the abovedescribed gradient plate procedure test against methicillin-resistantStaphylococcus aureus, (in the absence of blood serum/in the presence of20 percent blood serum).

7-m-Chlorophenylthioacetamidocephalosporanic acid, potassium salt,(2.0/5.2) 7-[2-(2",5"-dichlorophenylthio)acetamido1-3-methyl-3-cephem-4-carboxylic acid,7-[2-(3",5"-dichlorophenylthio)acetamido]cephalosporanic acid, sodiumsalt, (2.0/8.7) 7[2'-(2,4"-dichloro-5"-fluorophenylthio)acetamido]cephalosporanic acid sodium salt, (3.0/ 20.0)7-[2"(3",4-dichlorophenylthio)acetamido1cephalosporanic acid, sodiumsalt, (1.0/1.7)7-[2'-(2",5"-dichlorophenylthio)acetarnido]cephalosporanic acid, sodiumsalt; (0.8/1.0) 3-carbarnyloxymethyl-7-[2-(2",5"-

dichlorophenylthio)acetamido]-3-cephem-4- carboxylic acid (4.1/1.7)

The selected halophenylthioacetamidocephalosporanic acid can be refluxedwith pyridine at 60C. for hours'to form the corresponding3-(N-pyridinomethyl) compound which can be recovered from the reactionmixture by procedures analogous to those described, for example, in U.S.Pat. No. 3,449,338 and 3,577,412. Theselectedhalophenylthioacetamidocephalosporanic acid can be converted tothe corresponding 3- azidomethyl-compound by heating an organic solventsolution thereof with sodium azide at a pH of about 7, maintained with asuitable buffer such as disodium phosphate. Similarly, the selectedhalophenylthioacetamidocephalosporanic acid solution can be heated withsodium d ithiocarbamate to form the corresponding3-aminothiocarbonylthiomethylcompound, by procedures analogous to thosefound in the literature, e.g., in U.S. Pat. No. 3,573,298.

Compounds having the 3-methyl side chain can be prepared by acylating7-aminodesacetoxycephalosporanic acid, (7-ADCA) disclosed in U.S. Pat.3,124,576. They can also be prepared by acylating 7-aminodesacetoxycephalosporin esters obtained by penicillin sulfoxid'e esterrearrangement as taught, e.g. in Hatfield U.S. Pat. No. 3,591,585.

The compounds having the thiomethyl, S-methyl- 1,3 ,4-thiadiazol-2'ylthio, 1 -methyl- 1 H-tetrazol- S-ylthio, 5-phenyl-1,3,4-oxadiazol-2-ylthio, 1H- tetrazol5-ylthio,5-(p-nitrophenyl)-1,3,4-oxadiazol- 2-ylthio, or 5-( p-methoxyphenyl l,3,4-oxadiazol- 2-ylthio side chain in the 3-position can be prepared byreacting Cephalosporin C, or 7-aminocephalosporanic acid or ester andN-protected forms of such compounds with the appropriate thiolsaccording to thiolation procedures now known. See, for example, U.S.Pat. Nos. 3,516,966 and 3,530,123. The aminoadipoyl group can be cleavedfrom Cephalosporin C and the 7-amino group of the thiolated nucleus acidor ester can be acylated as described above. Ester groups are removed byreduction, hydrogenolysis or acid cleavage methods as appropriate forthe particular ester group, and salts of the acids are formed, ifdesired, to assist getting the desired compound out of its reactionmixture, and to increase solubility of the compound in the selectedpharmaceutical vehicle, if necessary. Acetoxymethyl esters of thehalophenylthioacetamido cephalosporanic acids may be used to enhanceabsorbability of the compound.

The following detailed examples exemplify proce- (lures for preparingthecompounds used in the method of this invention.

EXAMPLE 1 To a solution of 7.1 g (30 mM) of 3,4dichlorophenylmercaptoacetic acid 'in 100 ml dry benzene was added 7.5g(5.0 ml) (60mM') oxalyl chloride and onejdrop .ofDMF. This solutionwas'stirred 3 hrs at 25C. The benzene was then removed 'on a rotaryevaporator'resulting'in a yellowsyrup which was taken 1 to a cold's'olution 5c) of 8.5 g of 7-ACA in 200 ml.

50% aqueous acetone containing 8.5 g sodium bicarbonate. This mixturewas stirred for 2 hrs while slowly warming up to 25C. The acetone wasthen removed on a rotary evaporator, and the aqueous solution resultingwas layered with 100 ml ethyl acetate. Hydrochloric acid was added to pH2, and the layers mixed and separated. The aqueous phase wasre-extracted with two 50 ml portions of ethyl acetate, and the organicphases were combined and dried over magnesium sulfate. The organicsolution was filtered and evaporated on a rotary evaporator yielding aclear yellow syrup which was taken up in methanol (250 m1). To thissolution was added 30 mM sodium-Z-ethyl-hexanoate in 100 ml ethanol. Theresulting solution was chilled and reduced in volume to give whitecrystals of the sodium salt of 7- (3,4-dichlorophenylthioacetamido)cephalosporanic acid. The product had amaximum in its ultraviolet absorption spectrum at 258 my. (c=l 3,400)and exhibited a 1760 cm band in its infrared spectrum.

Note: DMF is N,Ndimethylformamide.

EXAMPLE 2 7-(2,5-Dichlorophenylthioacetamido)-cephalosporanic acid wasprepared as the sodium salt according to the procedure described inExample 1 and under the same conditions. The product had a maximum inits ultraviolet absorption spectrum at 253 my. (e -12,550) and exhibiteda 1760 cm band in its infrared spectrum.

EXAMPLE 3 7-(3,5-Dichlorophenylthioacetamido)-cephalospo ranic acid wasprepared as the sodium salt according to the procedure described inExample 1 and under the same conditions. The product had a maximum inits ultraviolet absorption spectrum at 260 mu (Fl5,600) and exhibited a1760 cm band in its infrared spectrum.

EXAMPLE 4 7(2,4-Dichloro-5-fluoro'phenylthioacetamido)cephalosporanicacid was obtained as the sodium salt ac cording to the proceduredescribed in Example 1 and under the same conditions. The product had amaximum in its ultraviolet absorption spectrum at 257 mu (F17,50O)andexhibited a 1760 cm band in its infrared absorption spectrum.

EXAMPLE 5 7-(3,4-Dichlorophenylthioacetamido)cephalosporanic acid sodiumsalt, 4.34 g., was dissolved in 100 ml aqueous sodium phosphate pH 7buffer. To this solution was added 1.16 g l-methyl-5mercaptotctrazole.This mixture was then heated at C for 4 hrs. The solution was thencooled, layered with ethyl acetate, acidified with HCl to pH 2, and thephases separated. The organic phase was dried over magnesium sulfate,filtered and reduced to a foam. This was taken up in ethano], anddicyclohexylamine added whereupon the product crystallized as thedicyclohexylammonium salt. The product of 7-[2-(3,4- dichlorophenylthio)acetamido]-3-( 1-methyltetrazol-5- yl-thiomethyl)-3 cephem-4-carboxylicacid, had a maximum in its ultraviolet absorption spectrum at 260 mu (Fl1,600) and exhibited a 1760 cm band in its infrared spectrum.

EXAMPLE 6 Following the procedure outlined in Example 5, 3.8 g of2,5-diehlorophenylthioacetamido cephalosporanic acid as sodium salt and1.1 g 5-methyI-1,3,4- thiadiazoIe-2-thiol were dissolved in 100 mlaqueous pH 7 phosphate buffer and heated at 70C for 4 hrs. The solutionwas Cooled, layered with ethyl acetate, and HCI added to adjust to pH 2.The organic layer was separated, dried over magnesium sulfate, andreduced to a gum. This gum was taken up in ethanol and the product,7-[2-(2,5-dichlorophenylthio)acetamidol-3-(5methy1-l,3,4thiadiazole-2-yl-thiomethyl)-3- cephem-4-carboxylic acid,subsequently crystallized as the acid upon standing. The product had amaximum in its ultraviolet absorption spectra at 254 mp. (e=15,200) andexhibited a 1700 cm band in its infrared spectrum.

EXAMPLE 7 571 mg 2,5-dichlorophenylthioaeetie acid was dissolved in 75ml methylene chloride, and to this solution was added 0.31 ml oxalylchloride and 2 drops DMF while the temperature was kept at to C for 1hr. The solution was reduced in volume on a rotary evaporator andsubsequently taken up in ml acetone. 546 mg7-Amino-3-(Nmethylcarbamoyloxymethyl)-A eephalosporanic acid wasdissolved in ml water containing 605 mg NaHCO Upon solution 5 ml acetonewas added and the solution was chilled to -5C. The acid chloride wasthen added dropwise to the cephalosporin nucleus and stirred 30 min at0C. The

fluorophenylthioacetamido)eephalosporanie acid, sodium salt was preparedby acylating 7-aminocephalos poranic .acid with3-chIoro-4-fluorophenylmercaptoacetic acid substantially according tothe procedure described in Example I. The product has a maximum in itsultraviolet spectrum at 254 millimicrons (e=10,400) and exhibited a 1760cm band in its infrared absorption.

EXAMPLE 9 This example compares the antibiotic activities of three knowncompounds, methicillin, cephalothin, and cephaloridine, with threecompounds from the above examples, in terms of minimum inhibitoryconcentration (MIC) values in microgram/milliliter units against variousisolates of Staphylococcus aureus cultures. The culture numbers and theprocedures used to obtain the reported data are disclosed by W. E. Wickand D. A. Preston in their article Heterogeneous Methicillin- ResistantStaphylococcus Aureus which appeared in Progress in Antimicrobial AndAnticancer Chemotherapy, Proceedings of the 6th International Congressof Chemotherapy, Volume 1, U. of Tokyo Press, Tokyo, (1970). In theseagardilution susceptibility tests, the inocula were diluted to 10bacteria per spot. Cultures 3055 (benzylpenicillin sensitive) and H232(benzylpenicillin-resistant) are controls. The remaining cultures aremethieillin-resistant, and, except for 3 136, are clinical isolates.Culture 3136 is a 4th transfer laboratory selected mutant. The resultsare given in the following table.

. AGAR DILUTION MIC PHENYL'IHIOACETYI. CEPHALOSPORINS vs METHICILLINRESISTANT S. uurcux MIC (pug/ml) lor Stupliylm'tu'cux uurvux cultures"Controls (Seattle) Kavarmak ANTIBIOTIC 3055 H232 3130 3131 3132 31333134 3135 3l36 3137 3138 313) 966 1138501 S61 Melhieillin" l 2 X X X 816 X X I28 I6 8 X X Ii Cephalothin .25 .25 It I 2 2 8 2 2 64 X l I 2Cephaloridine .06 .06 4 I 2 4 X I 0.25 X X I 0.25 l 4 Example 3 .06 .060.5 05 0.25 I 05 0.5 0.25 2 2 0.5 0.25 0.5 0.5 Example I .06 06 05 I 050,5 I 0.5 (L5 2 l l 0.5 0.5 0.5 Example 2 ()6 .06 0.5 0.5 0.5 I I 0.5 052 I I 0.5 0.5 0.5 Methicillin" 2 2 123 I28 32 I28 64 64 6-1 I2I I2I I2X64 I28 I28 Ccphalothin 25 25 64 32 I6 64 64 32 32 I28 64 32 32 32 64'(ephaloridine 0.25 8 I6 2 I6 16 8 I6 32 I6 4 0.25 4 l6 Example 3 ,06 .06l 2 0.5 I I I 2 4 I I 0.5 2 2 Example I 06 06 I I 0.25 I 0.5 0.25 0.5 20.5 0.25 0.25 0.5 Example 2 .06 .06 2 I 0.5 2 2 I I 4 I 0.5 0.25 I IFootnotes:

inocula diluted lo 10" hacleria per spot. "'Agur medium eonmins 0.5;sodium chloride. "'Agar medium contains 5.0% sodium chloride.

EXAMPLE 8 The compound 7-(3 '-chloro-4- These data show that thecompounds of Examples 1, 2 and 3 are substantially more effective thanmethicillin, cephalothin, and cephaloridinc in in vitro tests againstvarious cultures of methicillin-resistant Staphy- [OCOCCHS aureus.

EXAMPLE 10 This example compares the antibiotic activities ofmethicillin, cephalothin and eephaloridine with the compounds ofExamples 1, 2 and 3, in broth dilution tests. The MIC values, in ,ug/ml,are against the same Staphylococcus aureus cultures described in Example9, except that in this example the cultures were grown in trypticase soybroth (TSB). Growing the cultures in two broth cultures, one having alow (0.5 percent) so- Med. 1.,43 (suppl); 40-46. One expects thatsimilar 1 changes in MIC values for this type of culture to other BROTHD11.

tures are susceptible to the cephalosporin antiobiotics used in thisinvention.

EXAMPLE 11 This example exemplifies the antibiotic activity of 7- [2-( 3'-chloro-4"-fluororphenylthio )a'cetamido1cephalosporanic acid, a watersoluble compound, described in Example 8, against various Staphylococcusaureus cultures, in a modified Bauer-Kirby Disc Susceptibility Test(Bauer, A. W., W. M. M. Kirby, J. C. Sherris; and

UTlON MlC PHENYLTHIOACETYI. CEPHALOSPORlNS vs METHlClLLlN RESISTANT S.uuruux Ml( (mg/ml) for Slap/1ylm'uccm' uurcus cultures ExperimentControls 1.

. I (Seattle) Kavarnick Antibiotic 3055 3130 3131 3132 3133 3134 31353136 3137 3138 3139 .966 1138501 S35 $61 Methicillin" 2.0 4.0 16 16 32 832 32 32 128 128 16 8.0 32 16 16 Cephalothin 0.5 0.5 8 2.0 2.0 2.0 322.0 2.0 64 32 4.0 2.0 4.0 8 8 Cephaloridine ND ND ND 2.0 2.0 1.0 ND 2.05 8 4.0 1.0 4.0 2.0 4 4 Example 3 .06 0.12 .5 .5 .5 .5 1.0 .5 0.5 2.01.0 2.0 1.0 0.5 0.5 0.5 Example 1 .06 0.12 0.5 1.0 .5 .5 1.0 .5 0.5 2.01.0 1.0 0.5 0.5 2.0 2.0 Example 2 .06 0.12 1.0 .5 .5 .5 2.0 .5 0.25 2.02.0 0.5 0.5 0.5 0.5 0.5 Methicillin" 2.0 4.0 64 128 64 128 64 64 128 128128 128 64 128 16 64 Cephalothin 0.5 0.5 64 64 64 64 64 64 64 128 64 3232 64 64 64 (.ephaloridine ND ND ND 16 16 16 ND l6 16 32 16 8 8 16 16 16Example 3 .06 0.12 2.0 1.0 1.0 2.0 2.0 2.0 2.0 4.0 2.0 2.0 8 1.0 2.0 1.0Example 1 .06 0.25 4.0 .0 2.0 2.0 4.0 2.0 2.0 4.0 1.0 1.0 2.0 2.0 2.02.0 Example 2 .06 0.25 8.0 2.0 2.0 2.0 4.0 2.0 4.0 4.0 2.0 0.5 2.0 2.02.0 2.0

Footnotes "lnocula of bacteria per ml. (ultures 3055themylpenieillin-sensitive1 and H232 for 3136. are natural isolates.Culture 3136 is a 4th transfer mutant.

'"I'SB hroth containing 0.51 sodium chloride.

' 1S0 broth containing 5.0; \tidIUttt chloride.

1hem lpenicillinresistant1 are controls. llie rest aremelhicillin-resistunt. and except B-lactam antibiotics, e.g., variouspenicillins and ceph- Turck, 1966 Amer-1 Clin- Pathol- In this test thecultures were incubated at both 37C. or 30C. According to the suggestionof D. I. Annear in his article entitled The Effect of Temperature onResistance of Staphylococcus aureus to Methicillin and Some OtherAntibiotics in The Medical Journal of Australia, Mar. 16, 1968, theheterogeneity of methicillinresistant S. aureus is manifested byincubation at 30C. By this method, the soluble 7-[2'-(3-chloro-4"-fluorophenylthio)acetamido]cephalosporanic acid invention, That is,methicillin-resistant S.aureus cul- (Example 8) appears more effectivethan cephalothin.

Bauer-Kirby Disc Susceptibility Test Example 8 Example 3 Ccphalothin S.uun'ux culture 30 mg Disc Change 30 mg Disc Change 30 mg Disc- Change37-30 3730 37-30 37 30 1mm) 37 30 (mm) 37 30 (mm) 3125 33.8 36.0 +2.234.2 37.0 +2.8 33.4 33.8 0.4 3130 33.2 31.0 -2.2 32.0 33.6 +1.6 32.820.2 -12.6 3131 26.6 29.0 +2.4 30.6 31.8 +1.2 29.6 16.6 -13.0 3132 29.428.0 1.4 31.0 32.6 H .6 28.6 17.6 i 11.0 3133 27.6 24.8 2.8 29.0 30.8{11.8 28.8 12.8 -16.0 3134. 28.0 27.8 +0.2 29.8 33.0 13.2 27.8 12.1 l5.73135 28.2 26.2 2.0 28.3 2 1.6 1 1.3 28.0 11.8 -16.2 3136 35.0 31.0 4.035.0 20.8 -15.2 35.6 20.8 -14.8 3137 24.4 22.1 -2.3 26.2 26.0 0.2 17.8 017.8 3138 25.6 25. 0.4 27.0 29.4 +2.4 I 26.2 11.3 14.9 313) 34.1) 31.6-2.4 36.0 32.2 3.8 32.0 27.0 5.0 3140 28.4 26.4 -2.0 28.8 30.2 +1.4 29.710.4 13.3 3055 35 35 35 35 35 35 3074" 36.0 36.0 0 35.0 35.0 0 31.8 32.1-1- 0.3 H232 33.4 34.3 40.) 34.1 32.6 -l.5 33.4 33.2 0.2

l-oolnotes.

31254140 Methicillin-resistant 1th \arying degrees of resistance tocephalothin. MISS-penicillin clisithe: 074. 11232-met1neil1m.\L.'|\.\111 'L[10110211110082pHKlUClflg.

EXAMPLE l2 1 The compound 7-[2-(3"-chloro-4"-fluorophenylthio)acetamido]cephalosporanic acid, prepared as describedin Example 8, was tested for antibiotic effectivenessagainstStreptococcus pyogenes, strain C203 infections in mice, and is comparedwith t the effectiveness of cephaloridine in the same test. Ex-

perience has shown with other. compounds, some of which are nowcommercial antibiotics, that a compound effective against Streptococcuspyogenes, strain C203 in this test will be effective againstStaphylococcus aureus strains in clinical use. This is intended asfurther evidence that the compounds of the method of this inventionshould be effective antibiotics in clinical situ- 1 upon crystals of theproduct appeared. The solid was filtered, washed with ethanol and driedin vacuo. The product exhibited a 1760 cm "band in the infrared spectrum(fi-lactam) and had an ultraviolet spectrum of 270 m (e 14,200). Allother hysica] data were ations. p

in this test, a standard invivo test method described F i t ptroposed is Z T TSS by Wick. w. 15.. F. Streightoff, and 1). 11. Holmes. a er f ra2 grea 196i, in J. BacterioL, 81: 233-235 was used. The oral P t 5 andsubcutaneous median effective doses (ED neci T 6 so u lty 0 d 6 f g il f4 essary to cure 50 percent of the mice were calculated n e h n f i ti3d 1 3 (5 l'igpi according to the method of Reed and Muench (Reed, 3939n l T 1 h j j L. 1., and H. Muench. 1938. Am. J. Hyg. 27: 493497 y me ye f 'f.

acid, sodium salt was tested for its ant1b1ot1c effectwe- The resultswere as follows.

. ness against the same Staphylococcus aureus cultures infecting EDM)Challenge described in Example 9. The table which follows gives Ce halosorin Route (m /1 x 2 (XLD the minimum inhibitor concentration (MIC) 1nm1crop p s g y F I 8 O I v 7 27 9 grams of compound per milliliter ofculture (pg/ml), in e g 0 2| comparison with similar MIC values for theknown an- (ephaloridine Oral 0.323 27.9 tibiotics methicillin,cephalothin and cephaloridine Subcu 30 against the same strains ofbacteria.

Agar Dilution MIC Example 13 Compound vs. Methieillin ResistantStaphylococcus aureus MIC (pg/ml) for S. aureux cultures Controls v(Seattle) Kavar mak Agar Antibiotic 3055 3130 3131 3132 3133 3134 3135.3136 3137 3138 3139 966 11321501 S6] "l'rypticasc Methiciilin 1 2 4 4 42 x 4 4 x a 2 4 x 4 Soy Agar. (.ephalothin .13 2.5 .5 .5 .5 .5 2 l .5 21 .5 .5 1 1- 0.5% N:1(l Cephaloridine .03 .06 .5 .5 .25 .13 1 .5 .l3. 4l .5 .5 l 1 Example 13 .03 .06 .5 .25 .13 .13 1 .5 .25 1 1 .25 .25 .5 .5

Trypticase Melhicillin l l 32 I 8 2 32 32 64 64 32 l to 64 32 Soy AgarCephalothin .25 .25 l l .5 .5 l l l l 2' .25 .25 l l with 4.592('ephaloridine .l3 .l3 1 l .5 .25 l 4 4 4 4 .l3 .l3 8 l NaCl addedExample I3 .03 .06 .25 .25 .l3 .13 .25 5 .5 l .5 .03 .06 l .5

EXAMPLE 13 The compound of Example 13 gave the following in Synthesis ofvitro minimum inhibitory concentrations (MlC values), l r r r r 7-[2-l(3-chloro-4-fluorophenyl)thlo ]acetam1do]-3- g g agdmst the fol[[(5-methyl-l.3,4-thiadiazol-2-yl)thio]methyl1-3- g eephem-4-carboxylicacid, sodium salt. organism MIC (Lg/ml) I 3-Chloro4-fluorophcnylthioacetic. acld, 2.20 g. (10 Hum 3055 mm mM) wasdlssolved in dry benzene (50 ml.) and I0 ml. (penicillin G susceptible)of oxalyl chloride and 1 drop DMF was added to the so- 9 -9 0 (Pen G.resist.) lut1on. The reaction mixture was stirred 1 hour at 25 C.sjawh-s X66 400 i The benzene and excess oxalyl chloride was thenrefnwrgwiii. indolc Positive moved in vacuo to yield the acid chlorideas a yellowish i x' -l'z fzi i tlk a; syrup. This syrup was taken up inbenzene and again Ii. acrogcm'x 1917 I28 reduced in vacuo two times toremove any residual oxa- Q 64 C. jrcmulu CF17 128 lyl chloride and HCl.Th1s syrup was then taken up in (Wm-mm X239 123 20 ml. dry acetone andadded dropwise to a stirred sow- SE3 128 65 S. I \'phin1ur1'un1 128lution of 7-am1no-3-(5-methyl-l ,3,4-th1ad1azol-2-yl B. hnmcm-sepu-m 128thio methyl)-3-cephem-4-carboxylic acid, 3.44 g. (10 l..mlr-naer-arum Xl128 -Continued Organism MK tag/ml.)

1:. unrvlururu 64 C. lmpim/Lv A17 128 'l'. munIugruplrrlm 27 128 A.flm'us .2 I28 (I ulmi l l28 The MIC for the compound of Example 13against Streptococcus pyogenes C203 in broth was 0.008 ,ug/ml. The S.pyvgems subcutaneous median effective dose (ED value) was 0.281 ,ug/mlagainst a challenge dose of 1260 LD of the bacteria.

1 claim:

l. A ccphalosporin antibiotic compound of the formula chlorine selectedso that the phenyl ring is substituted with l or 2 chlorine atoms and sothat when one chlorine atom is present said chlorine atom is in the3-position, and when two chlorine atoms are present said chlorine atomsare in the 3,4-, the 3,5-, or the 2,5-positions; and,

when Z is fluorine, said fluorine is in the 3- or 4- position of thephenyl ring, and each of X and Y is hydrogen or chlorine selected sothat when the phenyl ring is substituted with 1 or 2 chlorine atoms. oneof the chlorine atoms is in the 3- or 4- position of the phenyl rings.

2. Compound of claim 1, in which Z is fluorine and each of X and Y ishydrogen: or chlorine selected so that the phenyl ring is substitutedwith l chlorine atom.

3. Compound of claim 1, in which Z is hydrogen.

4. Compound of claim 3, in which X and Y are both chlorine.

5. Compound of claim 4, in which the chlorines are in the 2,5-positions.

6. Compound of claim 4, in which R is l-methyl-ll-ltetrazol-S-ylthio.

7. Compound of claim 4, in which R is 5-mcthyll,3,4-thiadiazol-2-ylthio.

8. Compound of claim 4, in which R is lH-tetrazol- S-ylthio.

9. Compound of claim 2, in which the fluorine is in the 4-position.

10. Compound of claim 9, in which R isS-methyll,3,4-thiadiazol-2-ylthio.

11. Compound of claim 9, in which R is l-methyl-l H tctrazol-S-ylthio.

12. Compound of claim 9. in which R is lH-tetrazol- S-ylthio.

1. A CEPHALOSPORIN ANTIBIOTIC COMPOUND OF THE FORMUALA
 2. Compound ofclaim 1, in which Z is fluorine and each of X and Y is hydrogen orchlorine selected so that the phenyl ring is substituted with 1 chlorineatom.
 3. Compound of claim 1, in which Z is hydrogen.
 4. Compound ofclaim 3, in which X and Y are both chlorine.
 5. Compound of claim 4, inwhich the chlorines are in the 2,5-positions.
 6. Compound of claim 4, inwhich R is 1-methyl-1H-tetrazol-5-ylthio.
 7. Compound of claim 4, inwhich R is 5-methyl-1,3,4-thiadiazol-2-ylthio.
 8. Compound of claim 4,in which R is 1H-tetrazol-5-ylthio.
 9. Compound of claim 2, in which thefluorine is in the 4-position.
 10. Compound of claim 9, in which R is5-methyl-1,3,4-thiadiazol-2-ylthio.
 11. Compound of claim 9, in which Ris 1-methyl-1H-tetrazol-5-ylthio.
 12. Compound of claim 9, in which R is1H-tetrazol-5-ylthio.